Ref. Ares(2014)2078985 - 25/06/2014
GSW/1629
125-15
Recommendation from
the Scientific Committee on
Occupational Exposure Limits
for Aniline (Addendum 2014)
SCOEL/SUM/153
June 2014
Draft for 2-month consultation
Deadline: 1 September 2014
DRAFT for consultation, deadline 1 September
Employment, Social Affairs & Inclusion
SCOEL Recommendation on Aniline
Table of Contents
1. Substance identification, physico-chemical properties.......................................... 3
2. Occurrence ................................................................................................... 4
3. Health significance ......................................................................................... 4
3.1. Mode of action of spleen toxicity and carcinogenicity .................................... 4
3.2. Toxicokinetics .......................................................................................... 5
3.2.1. Biological monitoring ........................................................................... 5
3.3. Human experimental exposure ................................................................... 5
4. Recommendation ........................................................................................... 6
5. References .................................................................................................... 8
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SCOEL Recommendation on Aniline
Recommendation from the
Scientific Committee on Occupational Exposure Limits
for Aniline
(Addendum 2014)
8-hour TWA:
2 ppm (7.74 mg/m3)
STEL:
5 ppm (19.35 mg/m3)
BLV:
0.5 mg aniline/l urine (sampling: end of shift)
Additional categorisation:
SCOEL carcinogen group C
(carcinogen with a practical threshold)
Notation:
“skin”
The SCOEL recommendation of an OEL for aniline (SCOEL/SUM/153), dated August
2010, was based on older human volunteer data by Dutkiewicz and Piotrowski (1961).
This study provided the only data of this kind in 2009/2010 (when discussed by
SCOEL). The study was not performed according to modern standards. In the
meantime, new data have become available that allow a re-evaluation. The present
addendum is therefore focussed on recent publications from 2009 on. These
publications shed new light on the mechanisms of experimental spleen toxicity and
carcinogenicity, and on quantitative aspects of methaemoglobin induction by aniline in
humans, which are connected to OEL derivation. For other aspects, the reader is
referred to the SCOEL Recommendation dated 2010.
1. Substance identification, physico-chemical properties
Name:
Synonyms:
Molecular formula:
EC No.:
CAS No.:
Molecular weight:
Conversion factors:
(20 C, 101.3kPa)
aniline
aminobenzene, phenylamine
C6H5NH2
200-539-3
62-53-3
93.127 g/mol
1 ppm = 3.87 mg/m3
1 mg/m3 = 0.258 ppm
EU harmonised classification:
Acute Tox. 3
Acute Tox. 3
Skin Sens. 1
Eye Dam. 1
Acute Tox. 3
Muta. 2
Carc. 2
STOT RE 1
H301
H311
H317
H318
H331
H341
H351
H372
Aquatic Acute 1
H400
Toxic if swallowed
Toxic in contact with skin
May cause an allergic skin reaction
Causes serious eye damage
Toxic if inhaled
Suspected of causing genetic defects
Suspected of causing cancer
Causes damage to organs through prolonged or repeated
exposure
Very toxic to aquatic life
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2. Occurrence
Besides being a compound with distinct occupational use, there is low background
exposure in the general population. A cross-sectional population-based survey in
Bavaria/Germany showed detectable levels of aniline in 93.9 % of 1 004 persons
(Kütting et al 2009). According to previous data of this group (Weiss and Angerer
2002), the excretion of aniline in the general population was 3.5 µg/l urine (median;
upper 95th percentile: 7.9 µg/l).
3. Health significance
3.1. Mode of action of spleen toxicity and carcinogenicity
As discussed in SCOEL/SUM/153, aniline has experimentally induced spleen tumours
in rats. Aniline was categorised by SCOEL as a Group C carcinogen with a practical
threshold, because carcinogenic doses caused early effects on haematological
parameters, inflammatory reactions in the spleen and perturbations of iron
metabolism as a result of haemolytic anaemia. In the meantime, this has been further
substantiated.
Ma et al (2008) exposed male Sprague-Dawley rats subchronically to aniline (0.5
mmol/kg/day via drinking water for 30 days). This aniline treatment led to a
significant increase in splenic oxidative DNA damage, measured as 8-hydroxydeoxyguanosine and to up-regulation of OGG and NEIL1/2 DNA glycosylases in spleen.
A second study (Wang et al 2010) evaluated the potential contribution of haeme
oxygenase-1 (HO-1), which catalyses haeme degradation and releases free iron. Male
Sprague-Dawley rats were given 1 mmol/kg/day aniline in water by gavage for 1, 4,
or 7 days, and respective controls received water only. Aniline exposure led to
significant increases in HO-1 mRNA expression in the spleen (2- and 2.4-fold at days 4
and 7, respectively) with corresponding increases in protein expression, as confirmed
by ELISA and Western blot analysis. Furthermore, immune-histochemical assessment
of spleen showed stronger immune-staining for HO-1 in the spleens of rats treated for
7 days, confined mainly to the red pulp areas. The increase in HO-1 expression was
associated with increases in total iron (2.4- and 2.7-fold), free iron (1.9- and 3.5fold), and ferritin levels (1.9- and 2.1-fold) at 4 and 7 days of aniline exposure. The
data suggested that HO-1 up-regulation in aniline-induced splenic toxicity could be a
pro-oxidant mechanism, mediated through iron release.
A third study (Ma et al 2011) examined whether base excision repair (BER) enzymes
NTH1 and APE1 contribute to the repair of oxidative DNA lesions in the spleen.
Treatment conditions of rats were as in the preceding studies. The treatment led to
significant increases in NTH1- and APE1-mediated BER activity in the nuclear extracts
of spleen of aniline-treated rats compared to the controls. NTH1 and APE1 mRNA
expression in the spleen showed 2.9- and 3.2-fold increases, respectively. This was
supported by Western blot analysis. The findings pointed to a mechanism through
which NTH1 and APE1 may regulate the repair of oxidative DNA damage under
conditions of aniline-induced splenic toxicity.
A fourth study (Ma et al 2013) examined whether NTH1 and APE1 contribute to the
repair of oxidative DNA lesions in the spleen following aniline treatment. This was
identical to that in the preceding studies. The treatment led to significant increase in
NTH1- and APE1-mediated BER activity in the nuclear extracts of spleen of anilinetreated rats compared to the controls. NTH1 and APE1 mRNA expression in the spleen
showed 2.9- and 3.2-fold increases, respectively. This was confirmed by Western blot
analysis. The increased repair activity of NTH1 and APE1 was discussed as an
important mechanism for the removal of oxidative DNA lesions.
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In essence, new data are fully in line with the view that the (experimental)
carcinogenic effect of aniline is linked to a mode of action that is associated with a
threshold: oxidative stress is involved in the spleen, and in response to oxidative
stress DNA repair pathways are operative.
Thereby, the categorisation in SCOEL group C of carcinogens is further supported.
3.2. Toxicokinetics
The toxicokinetics of aniline have been previously described in detail
(SCOEL/SUM/153). In the meantime, a comparative study on percutaneous
penetration of a number of chemicals, including aniline, provided additional support
for the assignment of a “skin” notation (Korinth et al 2012).
3.2.1. Biological monitoring
New data on biological monitoring became available with the human experimental
exposure study described in Section 3.3.
3.3. Human experimental exposure
Käfferlein et al (2014) conducted a controlled human exposure study in volunteers. In
a first pilot section of this study, 4 male were exposed for 8 hours to 2 ppm airborne
aniline. This resulted in an increase of methaemoglobin levels up to 1.6 %, with a
plateau after 6 hours. The maximal excretion of aniline in urine after exposure was
306 µg/l. The following main study included 19 non-smoking persons (10 males, 9
females), which were exposed to 2 ppm aniline for 6 hours. The basal
methaemoglobin level prior to exposure was 0.72 ± 0.19 %. Following exposure, the
maximum methaemoglobin levels in blood was 2.21 ± 0.29 % (range: 0.8–2.07 %),
and aniline excretion in the urine was 168.0 ± 51.8 µg/l (range: 79.5 ± 418.3 µg/l).
After 24 hours, the mean level of methaemoglobin returned to the basal level
(0.65 ± 0.18 %). No significant differences between males and females were noted.
Details of the time course of methaemoglobin in blood during and after exposure are
shown in Figure 1; the time course of aniline excretion in urine is shown in Figure 2.
There was an elevation of the level of aniline-haemoglobin adducts, which depended
on the acetylator status (NAT2; slow or fast acetylators), as shown in Figure 3. By
contrast, the methaemoglobin levels and aniline excretion showed no significant effect
of the acetylator status.
Figure 1. Time course of methaemoglobin (Met-Hb) levels in blood of 19 volunteers during and
after experimental exposure to 2 ppm airborne aniline (Käfferlein et al 2014).
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Figure 2. Time course of aniline excretion in the urine of of 19 volunteers during and after
experimental exposure to 2 ppm airborne aniline (Käfferlein et al 2014)
Figure 3. Time course of the aniline-haemoglobin adduct in the blood of 19 volunteers during
and after experimental exposure to 2 ppm airborne aniline (Käfferlein et al 2014).
In essence, the new study of Käfferlein et al (2014) shows that a single exposure to
aniline for 6–8 hours leads to methaemoglobin levels, which are far below the level of
4–5 %, which is regarded as being critical (SCOEL/SUM/153). The data also confirm
that there will be no carry-over of elevated methaemoglobin to the next shift upon
daily repetitive exposure. As the individual maximal methaemoglobin level under the
given experimental conditions was 2.21 %, even a moderate physical activity will not
lead to exceeding the critical level.
4. Recommendation
Since the SCOEL Recommendation SCOEL/SUM/153 new data have become available,
which give rise to a re-assessment of the former Recommendation.
Studies into the mechanism of the experimental splenotoxicity and carcinogenicity in
rats (Section 3.1) have provided further confirmation for a catagorisation into SCOEL
group C of carcinogens (with a practical threshold). Therefore, it is now well
established that chronic splenotoxicity and subsequent carcinogenicity is a secondary
process following an increased breakdown of erythrocytes because of aniline-induced
methaemoglobenaemia. As outlined in SCOEL/SUM/153, it follows that avoidance of
excessive methaemoglobinaemia will protect against carcinogenesis in the spleen.
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The new experimental human exposure study by Käfferlein et al (2014) supersedes
the data of Dutkiewicz and Pietrowski (1961), which was the basis of the former
recommendation of SCOEL. According to the new study, an exposure to 2 ppm aniline
for 8 hours will not lead to elevation of methaemoglobin beyond critical levels, even if
a moderate workload is considered (Section 3.3). Therefore, an airborne level of 2
ppm aniline is recommended as OEL (8-hour TWA).
A comparison of the methaemoglobin levels reached under the conditions of the study
of Käfferlein et al (see Figure 1) and the critical methaemoglobin levels of 4–5 %
(SCOEL/SUM/153) shows that the recommended OEL of 2 ppm aniline implies an
inbuilt safety factor of about 2. Therefore, no additional uncertainty factor is needed to
compensate for possible additional human inter-individual variation.
As outlined by SCOEL (SCOEL/SUM/153), a STEL is preferred to
exposures with possible methaemoglobin formation. In view of the
aniline and the rapid decrease of methaemoglobin, an excursion
provide adequate protection (SCOEL/SUM/153). Applying the
approach of SCOEL, a STEL of 5 ppm is therefore recommended.
limit short-term
short half-life of
factor of 2 will
preferred value
In line with the previous argumentation (SCOEL/SUM/153), which is supported by
additional data (Section 3.2), a “skin”notation is warrented.
Also regarding biological monitoring, the new study study of Käfferlein et al (2014)
supersedes the study of Dutkiewicz and Piotrowski (1961). Considering the time
course of aniline in urine during and after exposure (Section 3.3, Figure 2), it ought to
be expected that an exposure to the proposed OEL of 2 ppm aniline, even under
conditions of moderate workload, will not lead to aniline excretion post shift higher
than 500 µg/l urine. Therefore, a new BLV of 0.5 mg aniline/l urine is recommended.
The present Addendum was adopted by SCOEL on Date Month Year.
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5. References
Dutkiewicz T, Piotrowski J (1961). Experimental investigations on the quantitative
estimation of aniline absorption in man. Pure and Applied Chemistry 3:319-323.
Käfferlein HU, Broding HC, Bünger J, Jettkant B, Koslitz S, Lehnert M, Marek EM,
Blaszkewicz M, Monsé C, Weiß T, Brüning T (2014). Human exposure to airborne
aniline and formation of methemoglobin: a contributions to occupational exposure
limits. Arch Toxicol, epub ahead of print.
Korinth G, Schaller KH, Bader M, Bartsch R, Göen T, Rossbach B, Drexler H (2012).
Comparison of experimentally determined and mathematically predicted
percutaneous penetration rates of chemicals. Arch Toxicol 86(3):423-430.
Kütting B, Göen T, Schwegler U, Fromme H, Uter W, Angerer J, Drexler H (2009).
Monoarylamines in the general population – a cross-sectional population-based
study including 1004 Bavarian subjects. Int J Hyg Environ Health 212(3):298-309.
Ma H, Wang J, Abdel-Rahman SZ, Boor PJ, Khan MF (2008). Oxidative DNA damage
and its repair in rat spleen following subchronic exposure to aniline. Toxicol Appl
Pharmacol 233(2):247-253.
Ma H, Wang J, Abdel-Rahman SZ, Hazra TK, Boor PJ, Khan MF (2011). Induction of
NEIL1 and NEIL2 DNA glycosylases in aniline-induced splenic toxicity. Toxicol Appl
Pharmacol 251(1):1-7.
Ma H, Wang J, Abdel-Rahman SZ, Boor PJ, Khan ME (2013). Induction of base excision
repair renzymes NTH1 and APE1 in rats spleen following aniline exposure. Toxicol
Appl Pharmacol 267(3):276-283.
Wang J, Ma H, Boor PJ, Ramanujam VM, Ansari GA, Khan MF (2010). Up-regulation of
heme oxygenase-1 in rat spleen after aniline exposure. Free Radic Biol Med
48(4):513-518.
Weiss T, Angerer J (2002). Simultaneous determination of various aromatic amines
and metabolites of aromatic nitro compounds in urine for low level exposure using
gas chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed
Life Sci 778(1-2):179-192.
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